Acidic biofilm remediation

ABSTRACT

Methods and compositions for biofilm remediation are disclosed. Biofilm is reduced and removed from soiled surfaces by providing to a surface in need an effective amount of a composition comprising a biocidal surfactant and at least one organic solvent. According to the invention, the biofilm remediation compositions reduce and remove biofilm formation by administering a one-step cleaner and disinfectant. The biofilm remediation compositions are stable and effective in concentrated and diluted ready-to-use formulations comprising an anionic sulfated or sulfonated surfactant that is not an organocarboxylic acid, a sparingly soluble organic solvent and optionally a soluble organic solvent.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Continuation application which claims priority tononprovisional application U.S. Ser. No. 16/110,821, filed Aug. 23,2018, which is a continuation of U.S. Ser. No. 13/045,734, filed Mar.11, 2011, now U.S. Pat. No. 10,085,447, issued Oct. 2, 2018, all ofwhich are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of biofilm remediation, including thetreatment of a variety of biofilm-soiled surfaces. More particularly,the invention relates to compositions, compounds, and methods forreducing and removing biofilm.

BACKGROUND OF THE INVENTION

Biofilms are biological conglomerates that contain pathogens, such asbacteria and other microorganisms, embedded in a matrix of exopolymersand macromolecules. In addition to bacteria, other microorganisms arecommonly found in biofilm, including fungi, molds, algae, protozoa,archaea and mixtures of these microorganisms. Biofilms form as a resultof microorganisms establishing on a surface and producing a protectiveextracellular polymeric matrix. Most often biofilm form on surfaces incontact with water, providing a hydrated matrix of polysaccharides toprovide structural protection from biocides, making biofilm moredifficult to kill than other pathogens.

Microbial infection and the formation of biofilm present significantcomplications in numerous industries. Although biofilm are known toexist in a wide-variety of environmental conditions, since biofilm mostoften form on surfaces exposed to bacteria and water, industries such asfood processing are commonly affected by biofilm. For example, theorganism Listeria monocytogenes thrives in cool, damp environments, suchas floor drains, plumbing and other surfaces of food processingfacilities. This provides a potential point of contamination for aprocessing plant environment and food products produced therein.However, biofilm can also develop on inert surfaces of everydayhousehold items. Exposure to such microorganisms through skin-surfacecontact may result in infections and compromise the public's health.Therefore, controlling the formation of biofilm is desirable to decreaseexposure to infectious microorganisms.

Biofilm growth and removal depends on several factors, including thesurface composition and chemical composition of the surroundingenvironment. Several biofilm removal methods are utilized, includingphysical, chemical and biological removal. Means of physically removingbiofilm include the use of magnetic fields, ultra sound, high and lowelectrical fields and abrasive techniques. Physical removal techniquesare often combined with chemical or biological methods, such as biocidesor antimicrobial agents. A number of technologies have been developedthat treat surfaces with organic or inorganic materials to interferewith biofilm development, such as preventing microbial attack anddegradation. For example, coating a surface with or incorporating acomposition into a surface substrate to create a surface whereinmicroorganisms do not adhere or colonize. U.S. patent application Ser.No. 12/134,353. However, such technologies have not effectivelyeliminated biofilm formation and growth. Therefore, the contamination ofsurfaces with biofilm remains a problem.

In light of the foregoing, there remains a demand for compositions andmethods for reducing and removing biofilm.

Accordingly, it is an objective of the claimed invention to developimprovements in biofilm remediation methods and compositions.

A further object of the invention is a method of biofilm remediationcapable of cleaning and disinfecting a variety of surfaces.

A further object of the invention is the development of compositions forthe effective remediation of biofilm.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention is a method for reducing and removingbiofilm by providing to a surface in need thereof an effective amount ofa composition including a biocidal surfactant and at least one organicsolvent at an acidic pH. In a preferred embodiment, the compositionincludes a biocidal anionic sulfated or sulfonated surfactant, asparingly soluble organic solvent, and optionally a soluble organicsolvent.

In another embodiment, the invention provides methods for reducing andremoving biofilm in or on a hard surface, which comprises providing thesurface in need thereof with an effective amount of one or more of thebiofilm remediation compositions. The biofilm remediation compositionsof the present invention reduce and remove biofilm formation byadministering a one-step cleaner and disinfectant. The biofilmremediation compositions are stable and effective in concentrated formsand diluted ready-to-use solutions.

According to the invention, the biofilm remediation compositions providesynergistic reduction in biofilm concentration at neutral pH as well aspH<7. Embodiments of the invention disclose the synergistic combinationof surfactant and solvent system to enable biocidal activity beyond theactivity of the surfactant agent in the compositions of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention pertains to the field of biofilm remediation. Moreparticularly, the invention provides novel methods and compositions forreducing and removing biofilm formation on various surfaces.

The embodiments of this invention are not limited to particular methodsand compositions for biofilm remediation, which can vary and areunderstood by skilled artisans. It is further to be understood that allterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting in any manner orscope. For example, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” can include pluralreferents unless the content clearly indicates otherwise. Further, allunits, prefixes, and symbols may be denoted in its SI accepted form.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which embodiments of the invention pertain. Many methods andmaterials similar, modified, or equivalent to those described herein canbe used in the practice of the embodiments of the present inventionwithout undue experimentation, the preferred materials and methods aredescribed herein. In describing and claiming the embodiments of thepresent invention, the following terminology will be used in accordancewith the definitions set out below.

The term “about,” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuring andliquid handling procedures used for making concentrates or use solutionsin the real world; through inadvertent error in these procedures;through differences in the manufacture, source, or purity of theingredients used to make the compositions or carry out the methods; andthe like. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

The term “alkyl,” as used herein, refers to a straight or branched chainmonovalent hydrocarbon radical having a specified number of carbonatoms. Alkyl groups may be unsubstituted or substituted withsubstituents that do not interfere with the specified function of thecomposition and may be substituted once or twice with the same ordifferent group. Substituents may include alkoxy, hydroxy, mercapto,amino, alkyl substituted amino, nitro, carboxy, carbanoyl, carbanoyloxy,cyano, methylsulfonylamino, or halo, for example. Examples of “alkyl”include, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, 3-methylpentyl, and thelike.

The term “antimicrobial,” as used herein, refers to the ability to killor inhibit the growth of microorganisms. According to the invention, thecompositions and methods of biofilm remediation in their broadest sensemay differ from current governmental regulations, such as regulationsfor antimicrobial standards. Therefore, the use in connection with thisinvention of the term “antimicrobial” and the like is not intended toindicate compliance with any particular governmental standard forantimicrobial activity.

The term “aryl,” as used herein, refers to monovalent unsaturatedaromatic carbocyclic radicals having a single ring, such as phenyl, ormultiple condensed rings, such as naphthyl or anthryl. Aryl groups maybe unsubstituted or substituted with those substituents that do notinterfere with the specified function of the composition. Aryl may besubstituted by halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₁-C₆ alkyl, C₁-C₆ substituted alkoxy, substituted C₂-C₆alkenyl, substituted alkoxy, amino, nitro, cyano, carboxy,hydroxymethyl, aminomethyl, carboxymethyl, C₁-C₄ alkylthio, hydroxy,C₁-C₄ alkanoyloxy, carbamoyl, or halo-substituted C₁-C₆ alkyl and may besubstituted once or more with the same or different group. Such an arylring may be optionally fused to one or more of another heterocyclicring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl rings. Examplesof “aryl” include, but are not limited to, phenyl, 2-naphthyl,1-naphthyl, biphenyl, 2-hydroxyphenyl, 2-aminophenyl, 2-methoxyphenyland the like.

The term “biofilm,” as used herein, means an extracellular matrix inwhich a population of microorganisms are dispersed and/or form colonies.Biofilms are understood to be typically made of polysaccharides andother macromolecules, often referred to as exopolysaccharides, that areconcentrated at an interface (usually solid/liquid) and act as a bindingagent that surrounds such populations of microorganisms. Biofilms arefurther understood to include complex associations of cells,extracellular products and detritus (or non-living particulate organicmaterial) that are trapped within the biofilm or released from cellswithin the biofilm. The term biofilm, as used herein, further refers tothe ASTM definition of biofilm as an accumulation of bacterial cellsimmobilized on a substratum and embedded in an organic polymer matrix ofmicrobial origin. Biofilms are understood to be a dynamic,self-organized accumulation of microorganisms and microbial andenvironmental by-products that is determined by the environment in whichit lives. According to the invention, the phrases “biofilm remediation,”“removing biofilm,” “reducing biofilm” and like phrases, shall mean theuse of the chemical biocide according to the invention which causes areduction in the rate or extent of biofilm growth, removal of existingbiofilm or portions of biofilm on surfaces and/or eradication ofexisting biofilm on a treated surface. According to the invention, thebiocidal compositions disclosed herein physically remove and killbiofilm.

The term “disinfectant,” as used herein, refers to an agent that killsall vegetative cells including most recognized pathogenicmicroorganisms, using the procedure described in A.O.A.C. Use DilutionMethods, Official Methods of Analysis of the Association of OfficialAnalytical Chemists, paragraph 955.14 and applicable sections, 15thEdition, 1990 (EPA Guideline 91-2).

The term “microorganism,” as used herein, refers to any noncellular orunicellular (including colonial) organism. Microorganisms include allprokaryotes. Microorganisms include for example, bacteria (includingcyanobacteria and Mycobacteria), lichens, microfungi, protozoa, virinos,viroids, viruses, and some algae. As used herein, the term “microbe” issynonymous with microorganism.

The term “sanitizer,” as used herein, refers to an agent that reducesthe number of bacterial contaminants to safe levels as judged by publichealth requirements. In an embodiment, sanitizers for use in thisinvention will provide at least a 99.999% reduction (5-log orderreduction). These reductions can be evaluated using a procedure set outin Germicidal and Detergent Sanitizing Action of Disinfectants, OfficialMethods of Analysis of the Association of Official Analytical Chemists,paragraph 960.09 and applicable sections, 15th Edition, 1990 (EPAGuideline 91-2). According to this reference a sanitizer should providea 99.999% reduction (5-log order reduction) within 30 seconds at roomtemperature, against several test organisms.

The term “sparingly soluble,” as used herein, refers to a second solventthat is soluble in a first solvent to an extent of less than about 20wt-%, preferably less than about 10 wt-%, preferably less than about 5wt-%, preferably less than about 3 wt-%. For example, a second solventthat is sparingly soluble in a first solvent can be soluble to an extentof 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 wt-%, to less than anyof these weight percentages, to any of these weight percentages modifiedby about, or to less than any of these weight percentages modified byabout.

The term “weight percent,” “wt-%,” “percent by weight,” “% by weight,”and variations thereof, as used herein, refer to the concentration of asubstance as the weight of that substance divided by the total weight ofthe composition and multiplied by 100. It is understood that, as usedhere, “percent,” “%,” and the like are intended to be synonymous with“weight percent,” “wt-%,” etc.

As used herein, the differentiation of antimicrobial “-cidal” or“-static” activity are definitions which describe the degree ofefficacy, and the official laboratory protocols for measuring thisefficacy are considerations for understanding the relevance ofantimicrobial agents and compositions. Antimicrobial compositions caneffect two kinds of microbial cell damage. The first is a lethal,irreversible action resulting in complete microbial cell destruction orincapacitation. The second type of cell damage is reversible, such thatif the organism is rendered free of the agent, it can again multiply.The former is termed microbicidal and the later, microbistatic. Asanitizer and a disinfectant are, by definition, agents which provideantimicrobial or microbicidal activity.

The biofilm remediation methods and compositions according to theembodiments of the invention present a significant improvement in theprior art and represent a significant change for industries in need ofcleaning and sanitizing products for biofilm. The biofilm remediationmethods and compositions according to the invention obviate the need fornumerous biofilm-reducing agents that are individually and/or incombination unable to completely remove and/or kill biofilm. The biofilmremediation compositions according to the invention provide a superiorbiocidal product, resulting in improved kill rates of biofilm over knownmethods of chemical and biological removal or reduction. This is abeneficial result of the biofilm remediation compositions according tothe invention having a “kill mechanism” capable of penetrating alllayers of a biofilm composition and reaching the substrate surface.These and other benefits of the biofilm remediation methods andcompositions according to the invention will be readily apparent basedon the description contained here, providing improved compositions andmethods for treating ubiquitous biofilm.

Various biofilm-reducing agents are known to provide some beneficialeffects in biofilm reduction and/or prevention. For example, chelatingagents such as EDTA and EGTA, chlorine, iodine and hydrogen peroxidehave previously been used as biofilm-reducing agents. Chelating agentsdestabilize the outer cell membrane of the biofilm. Chlorine, iodine,and hydrogen peroxide remove biofilm by depolymerizing the matrix.Further, biofilm-reducing agents may include antimicrobial proteins,such as nisin, which may be produced by Lactococcus lactus. Biocides orantimicrobial agents are also used as biofilm-reducing agents. Examplesof biocides or antimicrobial agents that are effective include:iodophores; phenols including halo- and nitrophenols and substitutedbisphenols such as 4-hexylresorcinol,2-benzyl-4-chlorophenol and2,4,4′-trichloro-2′-hydroxydiphenyl ether; quaternary ammonium compoundsand other cationic compounds; cationic surfactants such as alkyl andbenzyl quaternary compounds like N-alkyl (C₁₂-C₁₈) dimethylbenzylammonium chloride monohydrate, dimethyl didecyl ammonium chloride, andN-alkyl and (C₁₂-C₁₄) dimethyl I-napthylmethyl ammonium chloride;organic and inorganic acids and its esters and salts such asdehydroacetic acid, peroxycarboxylic acids, peroxyacetic acid, methylp-hydroxy benzoic acid; aldehydes such as glutaraldehyde; antimicrobialdyes such as is acridines, triphenylmethane dyes and quinones andhalogens.

However, as described according to the invention, the biofilmremediation compositions and methods described herein provide enhancedantimicrobial “-cidal” mechanisms that are superior over priorbiofilm-reducing agents. According to a preferred embodiment, thebiofilm remediation composition and methods provide up to a 5-log orderreduction in the population of microorganisms and pathogens in biofilm,compared to the optimal 3-log order reduction observed with use of thebiofilm-reducing agents described above. The beneficial results of thebiofilm remediation composition according to the invention result fromthe composition's penetration of all layers of a biofilm to thesubstrate surface, providing a complete kill of the microorganismshoused in such biofilm.

Compositions

Compositions according to the present invention were evaluated anddemonstrated to provide advantageous remediation of biofilm. Accordingto an embodiment of the invention, a biofilm remediation composition maycomprise from about 0.1 to about 90 wt-% of an anionic surfactant andfrom about 0.1 to about 60 wt-% of a sparingly soluble organic solvent.The biofilm remediation composition may further contain from about 0.1to about 60 wt-% of a soluble organic solvent. According to theinvention, the anionic surfactant is a sulfonated acid and thecomposition has a pH of less than about 5. According to a furtherembodiment of the invention, the anionic surfactant is a sulfonated acidand the composition has a pH of less than about 4, and preferably lessthan about 3.5.

According to another embodiment of the invention, the biofilmremediation composition may comprise from about 5 to about 90 wt-% of ananionic surfactant and from about 5 to about 50 wt-% of a sparinglysoluble organic solvent. The biofilm remediation composition may furthercomprise from about 5 to about 50 wt-% of a soluble organic solvent.According to this embodiment of the invention, the anionic surfactantmay be a sulfonated acid and the composition may have a pH of less thanabout 5. According to a further embodiment of the invention, the anionicsurfactant is a sulfonated acid and the composition has a pH of lessthan about 4, and preferably less than about 3.5.

According to a still further embodiment of the invention, the biofilmremediation composition comprises from about 10 to about 80 wt-% of ananionic surfactant, from about 15 to about 45 wt-% of a sparinglysoluble organic solvent, and optionally from about 15 to about 45 wt-%of a soluble organic solvent. According to this embodiment of theinvention, the anionic surfactant may be a sulfonated acid and thecomposition may have a pH of less than about 5. According to a furtherembodiment of the invention, the anionic surfactant is a sulfonated acidand the composition has a pH of less than about 4, and preferably lessthan about 3.5.

According to a further embodiment of the invention, a concentratedbiofilm remediation composition may comprise from about 0.5 to about 30wt-% of an anionic surfactant, from about 0.1 to about 10 wt-% of asoluble organic solvent and from about 0.5 to about 40 wt-% of asparingly soluble organic solvent. According to a preferred embodiment,a ready-to-use biofilm remediation composition may comprise from about0.2 to about 2 wt-% of an anionic surfactant and from about 0.5 to about5 wt-% of a sparingly soluble organic solvent in dilution.

According to a still further embodiment of the invention, a dilutedready-to-use biofilm remediation composition comprises the concentratedcomposition of the anionic surfactant, sparingly soluble organic solventand soluble organic solvent, diluted with water. According to apreferred embodiment the dilution is about 5 parts water per 1 partconcentrated composition to about 128 parts water per 1 partconcentrated composition. According to a further embodiment the dilutionis from about 8 parts water per 1 part concentrated composition to about64 parts water per 1 part concentrated composition. According to a stillfurther embodiment the dilution is from about 32 parts water per 1 partconcentrated composition.

Surprisingly, the biofilm remediation compositions according to theinvention are efficacious against both gram positive and gram negativebacteria across a broad range of pH. According to an embodiment of theinvention, the biofilm remediation compositions provide gram positiveand gram negative sanitizing effects at pH ranges from about 0 to 10, 1to 9, 2 to 8, and preferably from about 3 to 7. According to a preferredembodiment, the pH of a biofilm remediation composition is less thanabout 7, less than about 6, less than about 5, less than about 4, lessthan about 3.5, less than about 3, less than about 2 or less than about1.

Surfactants

Surfactants are incorporated in the present biofilm remediationcompositions. According to the invention, the biofilm remediationcomposition comprises a surfactant providing biocidal activity.According to a preferred embodiment of the invention, the biofilmremediation composition comprising a biocidal surfactant has a pH lessthan about 8. According to a preferred embodiment, the biofilmremediation composition has a pH of less than about 7, preferably lessthan about 6, more preferably less than about 5, and still morepreferably a pH between about 1 and about 4. According to a mostpreferred embodiment, the biofilm remediation composition comprising thesurfactant has an acidic pH between about 2 and about 4. Although notintending to be limited to a particular theory as to the nature of thecleaning and disinfecting properties of the biofilm remediationcomposition, the acidic pH of the composition comprising the surfactantpromotes the protonation of the surfactant to provide biocidal activity,rather than detergent activity in alkaline conditions.

Suitable surfactants for use in the acidic biofilm remediationcomposition of the invention, include anionic surfactants orsurface-active agents, sulfated surfactants or surface-active agents,sulfonated surfactants or surface-active agents and/or any combinationsthereof. The surfactant according to the invention may be a singlesurfactant or surface-active agent or combinations of surfactants and/orsurface-active agents.

Anionic surfactants may include, for example, sulfonates and surfaceactive sulfonates, such as sulfonated acids, and sulfates. Suitablesulfonates may include, for example, alkylsulfonates,alkylbenzenesulfonates, alkylarylsulfonates, diphenylated sulfonates,such as alkylated diphenyl oxide disulfonate, secondary alkanesulfonate, sulfonated fatty acid esters, sulfonated acids, and the like.Sulfonated acids may include, for example, xylene sulfonic acid,sulfonated oleic acid (also referred to as oleic acid sulfonates) andthe like. Suitable sulfates may include, for example, sulfated alcohols,sulfated alcohol ethoxylates, sulfated alkylphenols, alkylsulfates,sulfosuccinates, alkylether sulfates, alkyl sulfates, linear andbranched primary and secondary alkyl sulfates, alkyl ethoxysulfates,fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ethersulfates and the like. Numerous suitable surfactants according to theinvention are commercially available, for example, as Steppen PC-48™(sulfonated fatty acid methyl ester).

According to a further embodiment, suitable synthetic, water solubleanionic surfactants may include the ammonium and substituted ammonium(such as mono-, di- and triethanolamine) and alkali metal (such assodium, lithium and potassium) salts of the alkyl mononuclear aromaticsulfonates such as the alkyl benzene sulfonates containing from about 5to about 18 carbon atoms in the alkyl group in a straight or branchedchain, e.g., the salts of alkyl benzene sulfonates or of alkyl toluene,xylene, cumene and phenol sulfonates; alkyl naphthalene sulfonate,diamyl naphthalene sulfonate, and dinonyl naphthalene sulfonate andalkoxylated derivatives or their free acids. Suitable sulfonates includeolefin sulfonates, such as long chain alkene sulfonates, long chainhydroxyalkane sulfonates or mixtures of alkenesulfonates andhydroxyalkane-sulfonates. Suitable sulfonates include secondary alkanesulfonates.

According to a preferred embodiment, an alkylsufonate is the surfactant,preferably a (C₈-C₂₂) alkylsufonate surfactant, more preferably a(C₁₀-C₂₂) alkylsufonate surfactant. According to an alternativepreferred embodiment, the sulfonates surfactant is a linear alkylbenzene sulfonic acid (LAS) providing sufficient biocidal activity forbiofilm remediation and further solubilizing the sparingly solublesolvent of the composition. According to the invention, the surfactant,such as in a preferred embodiment using LAS is most effective at pH 3.0or below due to the protonated chemistry of the surfactant. In addition,the surfactant according to the invention is most effective incombination with the solvent components as described herein.

As will be apparent to those skilled in the art, the above-listedsurfactants are illustrative and various other surfactants meeting thecriteria set out above may also be used according to the invention.

According to an embodiment of the invention, the surfactants excludeshort chain (C1-C5) organocarboxylic acids. In particular, alpha- andbeta-hydroxycarboxylic acids, such as lactic acid and glycolic acid arenot selected as surfactants according to the biofilm remediationcompositions of the invention.

According to an embodiment of the invention, medium or long chainorganocarboxylic acids are used as surfactants for the biofilmremediation compositions. These include, but are not limited to C6-C10organocarboxylic acids, including for example, caproic, capric andcaprylic acid). Additional suitable medium or long chainorganocarboxylic acids include pelargonic and enanthic acids.

Soluble and Sparingly Soluble Organic Solvents

According to the invention, the biofilm remediation compositioncomprises at least one soluble and/or sparingly soluble organic solvent.The invention preferably comprises a two solvent biofilm remediationcomposition. The two solvent compositions preferably contain a firstsoluble organic solvent and a second solvent that is only sparinglysoluble in the first solvent. According to the preferred embodiment ofthe invention, the two solvent biofilm remediation composition firstcontains a diluting solvent (e.g., organic solvent). The soluble organicsolvent according to the invention is a hydrophilic (water soluble),miscible organic solvent. A suitable soluble organic solvent accordingto the invention keeps the composition's clarity and low viscosity toaid in forming and maintaining an easily dispensable biofilm remediationcomposition. According to a further embodiment, the soluble organicsolvent maintains a second sparingly soluble organic solvent insolution.

According to the preferred embodiment of the invention, the biofilmremediation composition further contains a sparingly soluble organicsolvent. According to an embodiment of the invention, the sparinglysoluble organic solvent aids the biocidal surfactant in penetrating themultiple biofilm layers, rather only penetrating a monolayer of atraditional non-biofilm soil. One skilled in the art can furtherascertain that the biofilm remediation composition may further compriseadditional solvents and/or cosolvents, such as alcohols. One skilled inthe art further understands that the solvents utilized according to theinvention do not degrade the biocidal surfactant or other solvents ofthe composition.

Suitable soluble organic solvents and sparingly soluble organic solventsaccording to the invention, include for example, glycol ethers or benzylalcohol. Although not intending to be limited according to a particulartheory for the effectiveness of the cleaning and disinfecting action ofthe biofilm remediation compositions, ether removes non-microbial soiland allows penetration of the biofilm layers to reach the substratesurface.

Suitable glycol ether solvents according to the invention, include, forexample, diethylene glycol n-butyl ether, diethylene glycol n-propylether, diethylene glycol ethyl ether, diethylene glycol methyl ether,diethylene glycol t-butyl ether, dipropylene glycol n-butyl ether,dipropylene glycol methyl ether, dipropylene glycol ethyl ether,dipropylene glycol propyl ether, dipropylene glycol tert-butyl ether,ethylene glycol butyl ether, ethylene glycol propyl ether, ethyleneglycol ethyl ether, ethylene glycol methyl ether, ethylene glycol methylether acetate, propylene glycol n-butyl ether, propylene glycol ethylether, propylene glycol methyl ether, propylene glycol n-propyl ether,tripropylene glycol methyl ether and tripropylene glycol n-butyl ether,ethylene glycol phenyl ether, propylene glycol phenyl ether and mixturesthereof. Numerous suitable solvents according to the invention arecommercially available, including for example, as DOWANOL EPh™ from DowChemical Co. (ethylene glycol phenyl ether), as DOWANOL DPM™ from DowChemical Co. (dipropylene glycol methyl ether) and DOWANOL PPh™ from DowChemical Co. (propylene glycol phenyl ether).

According to a preferred embodiment of the invention, glycol ethers areused as soluble and sparingly soluble organic solvents. According to amore preferred embodiment, the soluble organic solvent of the biofilmremediation composition is dipropylene glycol methyl ether and thesparingly soluble organic solvent is propylene glycol phenyl ether.According to a more preferred embodiment the soluble and sparinglysoluble organic solvents make a clear, low viscosity biofilm remediationcomposition upon dilution. A still further preferred embodiment of thecomposition according to the invention does not comprise anorganocarboxylic acid in the composition.

Oxidizing Agent

According to an embodiment of the invention, an oxidizing agent may befurther included in the biofilm remediation composition for biofilmremoval, cleaning and/or disinfecting microorganisms. The presentcomposition can include any of a variety of oxidizing agents, forexample, hydrogen peroxide. Additional suitable oxidizing agentsaccording to the invention, in addition to hydrogen peroxide, includefor example, inorganic and organic peroxides, such as, salts ofperborate, percarbonate, persulfate, perphosphate, persilicate,percarbonic acid, ozone and mixtures of the same.

According to a preferred embodiment, hydrogen peroxide or a hydrogenperoxide constituent may be included as an inorganic oxidizing agent.Hydrogen peroxide is commercially available at concentrations of 35%,50%, 70%, and 90% in water, with the 35% being most commonly used. Thepresent biofilm remediation compositions can include, for example, fromabout 0.01 wt-% to about 35 wt-% hydrogen peroxide or oxidizing agent,preferably from about less than 35 wt-% for concentrated biofilmremediation compositions and from about less than 5 wt-% forready-to-use biofilm remediation compositions.

Water and Additional Agents

The compositions of the invention may further include water. Water maybe present in the biofilm remediation composition from about 0.01 wt-%to about 90 wt-%. According to the invention, the compositions mayfurther comprise additional agents or adjuvants, such as aestheticenhancing agents (i.e., dye, perfume, etc.) and the like. Adjuvants andother additive ingredients will vary according to the preciseformulation of the biofilm remediation composition and its intendedmethods of use and can be included in the compositions in any amount.

Methods of Making Biofilm Remediation Composition

The composition according to the embodiments of the invention can beprepared by conventional manufacturing processes and equipment. Thecompositions can be dispensed in packaging typically utilized forcleaning products. Additional variations and modifications of theembodiments of the invention will be readily ascertainable to thoseskilled in the art.

Methods of Biofilm Remediation

In an embodiment of the invention, the methods include applying thebiofilm remediation compositions to a surface to reduce and/or eliminatebiofilm. A method for biofilm remediation can include contacting asurface with the biofilm remediation composition. For example, thecomposition can be introduced to a hard surface in contact with biofilm,such as walls, floors, sinks, countertops and commercial food servicesurfaces According to the invention, the biofilm remediation methodsinhibit biofilm by reducing and/or eliminating biofilm growth producedby a wide variety of bacteria and other microorganisms. For example,according to an embodiment of the invention, the methods for biofilmremediation are effective for biofilm comprising a variety of pathogens,such as both gram positive and negative bacteria, including for examplePseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis,Staphylococcus aureus and Listeria monocytogenes.

According to the invention, the biofilm remediation compositionsdescribed herein may be provided to a surface in contact with biofilm,by the application of the composition by wiping, spraying, dripping orthe like. After applying the biofilm remediation composition to asurface, the composition can be allowed to contact the biofilm-soiledsurface for a predetermined amount of time. The amount of time can besufficient to allow the composition to penetrate the biofilm matrix. Themethod may comprise a single step of applying the solution onto thesurface without direct physical removal, or may comprise bothapplication and removal steps in order to physically remove the biofilmfrom the treated surface.

According to an embodiment of the invention, the population of microbeson or in a biofilm-soiled surface is reduced. The biofilm remediationmethods of the invention provide greater than a 90% reduction (1-logorder reduction) in the population of one or more biofilmmicroorganisms, such as Pseudomonas. More preferably, the methodsprovide a greater than 99% reduction (2-log order reduction) in suchpopulation. In a preferred embodiment, the invention provides a greaterthan 99.9% reduction (3-log order reduction) in the population ofmicroorganisms and pathogens in biofilm. According to a more preferredembodiment, the invention provides a greater than 99.99% reduction(4-log order reduction) in such populations. A most preferred embodimentof the invention provides a greater than a 99.999% reduction (5-logorder reduction) in the population of microorganisms and pathogens inbiofilm.

According to the invention and the benefits described herein, thebiofilm remediation compositions and methods of using the same arefurther useful in reducing the opportunity for gene transfer withinbiofilm due to the high kill rates in such populations. As a result ofconjugation occurring at an increased rate in cells in biofilm, methodsproviding increased kill rates in the population of microorganisms andpathogens in biofilm, as demonstrated by up to a greater than 99.999%reduction (5-log order reduction), reduce gene transfer among suchpopulations. Although not intending to be limited according to aparticular theory, the methods of biofilm remediation according to theinvention effectively reduce biofilm resistance to antimicrobials orchemical biocides, specifically for bacterium, as bacterial remediationreduces gene transfer of resistance to neighboring susceptible bacteria.

Treated Surfaces

According to some embodiments, the methods and compositions of theinvention are applied to surfaces which are in need of cleaning and/ordisinfecting, including, but not limited to those surfaces described inU.S. Pat. No. 7,569,232. According to the invention, a variety of inertsurfaces where biofilm preferentially develops may be treated accordingto the methods of the invention, including surfaces of everyday andhousehold items. Examples of such surfaces include, but are not limitedto, hard surfaces, e.g., walls, floors, sinks, countertops andcommercial food service surfaces. Additional surfaces that may be inneed of cleaning and/or disinfecting according to the invention includesurfaces exposed to dampness or water, such as those found in waterand/waste treatment, such as drain lines, pipes and other plumbingsurfaces, tubes and valves and like. According to additional embodimentsof the invention, exemplary industries in which the methods andcompositions of the invention may be used include, but are not limitedto, institutional industries, including hotels, housekeeping andfoodservice; food processing; water care industries; janitorialindustries; and health care.

The methods of the present invention may be used for microbial control,e.g., to reduce or remove biofilm formation and/or for cleaning and/ordisinfecting of such surfaces. In certain embodiments, the biofilmremediation composition may be applied to the surface of a substratethat is susceptible to biofilm formation. The substrate may be made fromany material to which such composition according to the invention may beapplied. Representative examples of the kinds of materials from whichthe substrate may be made include porous materials, soft materials, hardmaterials, semi-hard materials, regenerating materials andnon-regenerating materials. According to preferred embodiments of theinvention, the substrate is a polymeric material (e.g., thermoplasticand thermoset), wood, metal, glass and ceramic, such as those materialsfound in the home and/or in public areas. Preferably, the substrate ismade from an inert material selected from the group consisting of apolymer, a metal, an alloy, and combinations thereof.

According to an embodiment of the invention, the methods of biofilmremediation are used on a variety of surfaces in need of such treatment.The biofilm remediation compositions may be frequently applied to suchsurfaces, including as often as necessary to completely remove biofilmpathogens. The biofilm remediation compositions are compatible with avariety of treated surfaces permitting daily use without being corrosiveto the treated surfaces. According to one embodiment, the biofilmremediation composition may be applied daily. As a result of the varioustreated surfaces being in contact with human tissue, for example in foodor drink products or preparation, the control of unwanted biofilmrequires the ongoing treatment of such surfaces and daily application,as permitted by the compatibility of the biofilm remediation compositionaccording to the invention, provides a beneficial treatment frequencyfor a variety of surfaces.

Foaming Applications

According to an embodiment of the invention, the biofilm remediationcompositions demonstrate enhanced surface retention time due to thefoaming profile of the composition. As a result, application of thecompositions according to the invention to a surface in contact with abiofilm can include administration of the composition to a drain orother surfaces in need of cleaning and/or disinfecting with a foamingchemistry. The biofilm remediation compositions are effective foamingchemistries suitable for administration into a trap to fill an entirevoid. A preferred embodiment of the invention is to administer thebiofilm remediation composition to a drain in order to reduce and/oreliminate biofilm growing in the drain.

According to the invention, the biofilm remediation compositionsdescribed herein may be provided to a surface in contact with biofilm,such as a drain, by filling the drain or other void. After applying thebiofilm remediation composition to the drain or other void, thecomposition remains in constant contact with the entire biofilm-soiledsurface for a predetermined amount of time as a result of its preferredfoaming profile. According to an embodiment of the invention, thebiofilm remediation composition contacts the entire contaminated surfaceof the treated surface, rather than only covering the limited areawherein a liquid would contact and flow through. The amount of time forthe interaction of the biofilm remediation composition with thebiofilm-soiled surface is selected to be an amount of time sufficient toallow the composition to penetrate the biofilm matrix.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1

A formulation was created by combining the components in the amountslisted in the table below for an embodiment of the biofilm remediationcomposition.

Component Wt-% Water 43.4 linear alkylbenzene sufonic acid 19.2dipropylene glycol methyl ether 5 propylene glycol phenyl ether 32.4

The removal of biofilm was tested to determine efficacy of biofilmremoval and kill rates of Pseudomonas aeruginosa. Pseudomonas arewell-known as common ‘pioneer’ bacteria and often tested forbiofilm-inhibiting agents' effectivity. The bacteria are known toexcrete polysaccharides and generate biofilm on a variety of surfacesvery rapidly (including, for example, stainless steel and polished hardsurfaces), as well as commonly demonstrate resistance to variousantimicrobial compositions. However, bacteria that exist in a biofilmare phenotypically different from suspended cells of the same genotype;therefore the study of biofilm in the laboratory requires protocols thataccount for this difference. Laboratory biofilms are engineered ingrowth reactors designed to produce a specific biofilm type. Alteringsystem parameters correspondingly results in a change in the biofilm.

Standard ASTM test methods for the quantification of a Pseudomonasaeruginosa biofilm grown with high shear and continuous flow using a CDCbiofilm reactor were followed (pursuant to ASTM Committee E-35 onPesticides and Subcommittee E-35.15 on Antimicrobial Agents). Thismethod was used to grow a repeatable Pseudomonas aeruginosa biofilm in acontinuously stirred flow reactor with high wall shear. The method alsoprovides instructions for sampling and analyzing biofilm for viablecells.

Pseudomonas aeruginosa (ATCC 700888) is the organism used. An isolatedcolony is aseptically removed from an R2A plate and placed into 100 mlof sterile bacterial liquid growth broth (300 mg/L) and incubated in anenvironmental shaker at 35° C.+2° C. for 20-24 hours. Viable bacterialdensity should equal 108 CFU/ml, and may be checked by serial dilutionand plating. Pseudomonas aeruginosa were grown in a CDC reactor systemfor 48 hours at room temperature. See Goeres, D. M., et al., Statisticalassessment of a laboratory method for growing biofilms, Microbiology151:757-762 (2005). Biofilm challenge is approximately 8 logs throughouttesting from a 48 hour growth.

Polycarbonate coupons were prepared. Coupons were removed from the rods,randomized and placed in containers where the biofilm remediationcompositions were applied to the coupons in the containers for thespecified exposure time (10 minutes). Coupons were then removed from thecontainer and placed in tubes containing neutralizer. The samples arevortexed, sonicated, serially diluted and plated for plate counts.Biofilm population density was recorded as log colony forming units persurface area.

Example 2

A biofilm remediation composition according to the invention anddisclosed below was tested for removal of biofilm and sanitizingefficacy was further tested to determine efficacy and kill rates againstStaphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 atboth 30 second and 2.5 minute exposure times. The tests demonstratesanitizing efficacy for inanimate, non-food contact surfaces.

Desired Test Solution (Volume of Test Concentration DiluentSubstance/Total Volume) pH 1:32 500 ppm 6.25 g/500 g total 2.11Synthetic Hard Water (pH 7.66) 6.99* *pH adjusted with 1.0N NaOH.

The test systems used an inoculum volume of 20 μL; dryingtime/temperature of 30-40 minutes at 35° C.; ambient testing temperature(18-22° C.); exposure time of 30 seconds and 2.5 minutes; neutralizermedia 20 mL Dey-Engley (DE) broth. A neutralizer screen was performed aspart of the testing, verified that the neutralizer adequatelyneutralized the product and was not detrimental to the tested organisms.Plating Medium utilized was Tryptone Glucose Extract (TGE) agar andbiofilm were incubated at 35° C. for 48 hours.

Results were analyzed according to the standard for non-food contactsanitizing efficacy screen (99.9% reduction in test organism growth isconsidered passing for a given exposure time).

Table 1 summarizes the average log growth of Staphylococcus aureus andPseudomonas aeruginosa observed after a 30 second exposure to 1:32biofilm remediation composition at its nominal pH and neutral pH 7.0.Test substances highlighted demonstrate reduction in the growth of S.aureus or P. aeruginosa by ≥99.9%, passing the non-food contactsanitizing efficacy screen.

TABLE 1 Average Standard Test Log₁₀ Deviation Percent Test SystemSubstance Survivors of Log Growth Reduction Staphylococcus 1:32 <1.400.00   100% aureus Nominal pH ATCC 6538 1:32 5.00 0.11 99.137% pH 7.0Pseudomonas 1:32 <1.40 0.00 99.999% aeruginosa Nominal pH ATCC 154421:32 <1.40 0.00 99.999% pH 7.0

Table 2 summarizes the average log growth of Staphylococcus aureus andPseudomonas aeruginosa observed after a 2.5 minute exposure to 1:32biofilm remediation composition at its nominal pH and pH 7.0 withhighlighted substances according to the invention reducing the growth ofS. aureus or P. aeruginosa by ≥99.9%.

TABLE 2 Average Standard Test Log₁₀ Deviation Percent Test SystemSubstance Survivors of Log Growth Reduction Staphylococcus 1:32 <1.400.00   100% aureus Nominal pH ATCC 6538 1:32 4.00 0.14 99.893% pH 7.0Pseudomonas 1:32 <1.40 0.00 99.973% aeruginosa Nominal pH ATCC 154421:32 <2.39 1.71 99.997% pH 7.0

The 1:32 dilutions of the biofilm remediation compositions according tothe invention demonstrate efficacious sanitizing results for allcompositions and treated biofilms at its nominal pH, as well as at a pHof 7.0 against Pseudomonas aeruginosa after both 30 seconds and 2.5minutes. In addition, at its nominal pH, a 1:32 dilution of the biofilmremediation composition passed against Staphylococcus aureus with a 30second and 2.5 minute exposure time. However, the results demonstrate anincrease in solution pH has a negative impact on efficacy at leastagainst S. aureus. At pH 7.0, the 1:32 dilution failed the non-foodcontact sanitizing screen after 30 seconds with a percent reduction ofonly 99.137%. Although the pH 7.0 passes the screen after 2.5 minutes,the percent reduction is 99.893%.

Example 3

A further biofilm remediation composition according to the invention anddisclosed below was tested for removal of biofilm and sanitizingefficacy was tested according to the conditions set forth in Example 2at a further dilution to determine efficacy and kill rates againstStaphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442 atboth 30 second and 2.5 minute exposure times. The tests demonstratesanitizing efficacy for inanimate, non-food contact surfaces.

Desired Test Solution (Volume of Test Concentration DiluentSubstance/Total Volume) pH 1:64 500 ppm 3.91 g/250 g total 2.51Synthetic Hard Water (pH 7.77) 6.98* *pH adjusted with 1.0N NaOH.

Results were analyzed according to the standard for non-food contactsanitizing efficacy screen (99.9% reduction in test organism growth isconsidered passing for a given exposure time).

Table 3 summarizes the average log growth of Staphylococcus aureus andPseudomonas aeruginosa observed after a 30 second exposure to 1:64biofilm remediation composition at a nominal pH (2.51) and a neutral pH(6.98). Test substances highlighted demonstrate reduction in the growthof S. aureus or P. aeruginosa by ≥99.9%, passing the non-food contactsanitizing efficacy screen.

TABLE 3 Average Standard Test Log₁₀ Deviation Percent Test SystemSubstance Survivors of Log Growth Reduction Staphylococcus 1:64 5.370.43 96.250% aureus Nominal pH ATCC 6538 1:64 5.80 0.25 89.968% NeutralpH Pseudomonas 1:64 4.18 1.12 98.079% aeruginosa Nominal pH ATCC 154421:64 4.65 0.41 94.277% Neutral pH

Table 4 summarizes the average log growth of Staphylococcus aureus andPseudomonas aeruginosa observed after a 2.5 minute exposure to 1:64biofilm remediation composition at its nominal pH and neutral pH withhighlighted substances according to the invention reducing the growth ofS. aureus or P. aeruginosa by ≥99.9%.

TABLE 4 Average Standard Test Log₁₀ Deviation Percent Test SystemSubstance Survivors of Log Growth Reduction Staphylococcus 1:64 1.600.35 99.999% aureus Nominal pH ATCC 6538 1:64 5.18 0.13 97.564% pH 7.0Pseudomonas 1:32 <1.40 0.00 99.997% aeruginosa Nominal pH ATCC 154421:32 4.63 1.13 94.545% pH 7.0

The 1:64 dilutions of the biofilm remediation compositions according tothe invention demonstrate the most efficacious sanitizing results for adiluted biofilm composition at its nominal pH against Pseudomonasaeruginosa and Staphylococcus aureus after 2.5 minute exposure.

Although not intended to be limited to a particular theory or mechanismof action, the biofilm remediation compositions according to theinvention demonstrate efficacious sanitizing results against both grampositive and gram negative bacteria at low pH (pH<5), according to thisExample at a pH 2.51. However, the 1:64 diluted biofilm remediationcompositions did not demonstrate the synergistic efficacy at a neutralpH—as achieved at the 1:32 dilution—as a result of the loss of activityfrom the combination of solvent and LAS. The dilution of the solventdemonstrates its partition into substrate (i.e. biofilm) occurring morequickly than the surfactant (showing a linear curve) even with dilutionof the compositions. However, regardless of the dilution of the solventsystem, the compositions demonstrate maintained efficacy against bothgram negative bacteria (typical of biofilm) and gram positive bacteriaprovides an unexpected result achieved by the biofilm remediationcompositions according to the invention.

Example 4

A non-food contact sanitizing efficacy test was conducted onready-to-use samples of 4-in-1 biofilm remediation composition againstPseudomonas aeruginosa ATCC 15442. A 2.5 minute exposure time wasutilized to test the sanitizing efficacy of compositions having aconsistent amount of LAS surfactant and compositions having a gradientof solvent in order to observe whether or not the LAS surfactantprovides the main source of efficacy. Table 5A shows the variation inthe biofilm remediation compositions having decreasing amounts ofsolvent in the formulations.

TABLE 5A R012111A R012111B R012111C R012111D Composition RM# Percentage% Water 100016 97.9800 98.1300 98.3300 98.4800 LAS 175075 0.6000 0.60000.6000 0.6000 DPM 164285 0.1600 0.1600 0.1600 0.1600 PPh 164048 1.00000.8500 0.6500 0.5000 NaOH (50%) 114132 0.2600 0.2600 0.2600 0.2600 Total100.0000 100.0000 100.0000 100.0000 pH 7.0 7.0 7.0 7.0

The test systems used an inoculum volume of 20 μL; dryingtime/temperature of 30-40 minutes at 35° C.; ambient testing temperature(18-22° C.); exposure time of 2.5 minutes; neutralizer media 20 mLDey-Engley (DE) broth. A neutralizer screen was performed as part of thetesting, verified that the neutralizer adequately neutralized theproduct and was not detrimental to the tested organisms. Plating Mediumutilized was Tryptone Glucose Extract (TGE) agar and biofilm wereincubated at 35° C. for 48 hours.

Table 5B shows the results for the 2.5 minute exposure time of thebiofilm remediation compositions having variations in solventconcentration.

TABLE 5B Test Average Log Geometric Percent Substance Rep CFU/mLCFU/Carrier* Survivors Mean Reduction 4-in-1 Cleaner 1 <1.0 × 10⁰  <2.5× 10¹  2.98  9.6 × 10² 99.893% 1.0% PPh 2 7.9 × 10¹ 1.98 × 10³  pH 7.0 39.2 × 10¹ 2.3 × 10³ 4 2.95 × 10²  7.38 × 10³  4-in-1 Cleaner 1 1.7 × 10³4.25 × 10⁴  4.58 3.84 × 10⁴ 95.728% 0.85% PPh 2 2.5 × 10³ 6.25 × 10⁴  pH7.0 3 1.3 × 10³ 3.25 × 10⁴  4 1.0 × 10³ 2.5 × 10⁴ 4-in-1 Cleaner 1 6.0 ×10⁴ 1.5 × 10⁶ 6.37 2.36 × 10⁶ 0.000% 0.65% PPh 2 6.0 × 10⁴ 1.5 × 10⁶ pH7.0 3 1.8 × 10⁵ 4.5 × 10⁶ 4 1.2 × 10⁵ 3.0 × 10⁶ 4-in-1 Cleaner 1 1.0 ×10¹ 2.5 × 10² 5.17 1.49 × 10⁵ 83.426% 0.50% PPh pH 7.0

Table 6 demonstrates the average log survivors of Pseudomonas aeruginosaATCC 15442 observed after a 2.5 minute exposure to 4-in-1 cleaners at pH7.0. The biofilm remediation composition with 1% solvent achieved areduction of P. aeruginosa by ≥99.9%, and therefore have passed thisnon-food contact sanitizing efficacy screen after 2.5 minutes. As shownin Table 6, a decrease in the percent reduction of P. aeruginosacorresponds with the decrease of solvent in the composition even with aconsistent level of LAS surfactant present.

TABLE 6 Average Standard Test Log₁₀ Deviation Percent Test SystemSubstance Survivors of Log Growth Reduction Pseudomonas 4-in-1 2.98 1.0899.893% aeruginosa 1.0% PPh ATCC 15442 4-in-1 4.58 0.17 95.728% 0.85%PPh 4-in-1 6.37 0.23 0.000% 0.65% PPh 4-in-1 5.17 1.86 83.426% 0.5% PPh

The variation in solvency supports the synergy of the biofilmremediation composition according to the invention. As the testedbiofilm remediation compositions in this Example were tested at aneutral pH were the base antimicrobial agent did not demonstrate atechnical effect. The gradient of solvent (PPh) in the formulationshowed at 1.0% a synergistic kill rate for the P. aeruginosa. Thetesting gradient further demonstrated a defined solvent effect lossmoving from the 1.0% solvent to the 0.5% solvent concentration.

Example 5

Foaming stabilization of the biofilm remediation composition wasanalyzed to assess its foaming profile. As shown in Table 7, the 4 in 1biofilm composition disclosed in Example 1 according to the inventionwas compared to various best in class foaming agents, includingPantastic, Scout and Oasis 135 (all commercially-available from Ecolab,Inc.) using the cylinder method. Initial foams for all products werespun for 4 minutes, using a 40 ml solution of each product.

TABLE 7 Foam Height (mls) Initial 2 5 7 10 Concen. Temp. Foam min- min-min- min- Product (oz/gal) (° F.) (mls) utes utes utes utes 4 in 1 1:32110 250 250 250 220 190 Pantastic 1:32 110 250 250 250 250 210 Scout1:32 110 250 250 250 250 250 Oasis 135 1:32 110 250 250 250 230 200

As demonstrated in Table 7, the biofilm remediation composition providescomparable foaming characteristics to commercially-available highfoaming products. The differences in foam height at 2 minutes, 5minutes, 7 minutes and 10 minutes show results that are equal to orcomparable to tested products. This foaming height achieved from thebiofilm remediation composition is suitable for use in filling voidswith constant contact of contaminated surfaces such as drain lines. Theability of a product to obtain this time of contact time in a void fillapplication directly leads to longer interaction of chemistry withbacteria on the surface. The foaming achieved by the biofilm remediationcomposition (and additional tested products) provides products havingboth an overall better coverage of surfaces as well as better cleaningand antimicrobial action as a result of the chemistries being in contactwith the surfaces for sufficient periods of time.

Although not intended to be limited to a particular theory of theinvention, the foaming activity of the biofilm remediation compositionis directly related to a solvent effect of the formulation. As a result,the application of the biofilm remediation composition in a foamingliquid base system allows the solvent to be added in a cost effectivemanner that is more advantageous to the environment.

The inventions being thus described, it will be obvious that the samemay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions and all suchmodifications are intended to be included within the scope of thefollowing claims.

1-20. (canceled)
 21. A concentrated biocidal composition comprising:from about 1 to about 30 wt-% of an anionic surfactant, wherein theanionic surfactant excludes short chain (C1-C5) organocarboxylic acidand is a sulfonated acid; from about 5 to about 25 wt-% of a solubleorganic solvent; and from about 10 to about 40 wt-% of a sparinglysoluble organic solvent; wherein said sparingly soluble organic solventis soluble in the soluble organic solvent to an extent of less thanabout 20 wt-%; wherein the pH of a diluted solution produced from theconcentrated composition is less than about 3; wherein the compositionprovides a 4-log order reduction of microbes in a biofilm-soiledsurface; and wherein the microbes comprise one or more of a Pseudomonas,an Escherichia, a Staphylococcus, and/or a Listeria.
 22. The compositionof claim 21 wherein the anionic surfactant is selected from the groupconsisting of (C₈-C₂₂) alkylsulfonate, linear alkyl benzene sulfonicacid, diphenylated sulfonates, sulfonated oleic acid, and mixturesthereof.
 23. The composition of claim 21 wherein said sparingly solubleorganic solvent is a glycol ether selected from the group consisting ofbenzyl alcohol, diethylene glycol n-butyl ether, diethylene glycoln-propyl ether, diethylene glycol ethyl ether, diethylene glycol methylether, diethylene glycol t-butyl ether, dipropylene glycol n-butylether, dipropylene glycol methyl ether, dipropylene glycol ethyl ether,dipropylene glycol propyl ether, dipropylene glycol tert-butyl ether,ethylene glycol butyl ether, ethylene glycol propyl ether, ethyleneglycol ethyl ether, ethylene glycol methyl ether, ethylene glycol methylether acetate, propylene glycol n-butyl ether, propylene glycol ethylether, propylene glycol methyl ether, propylene glycol n-propyl ether,tripropylene glycol methyl ether and tripropylene glycol n-butyl ether,ethylene glycol phenyl ether, propylene glycol phenyl ether and mixturesthereof.
 24. The composition of claim 23, wherein the sparingly solubleand soluble organic solvents are a mixture of dipropylene glycol methylether and propylene glycol phenyl ether.
 25. The composition of claim24, wherein the biocidal composition comprises between about 0.8 and 60wt-% of dipropylene glycol methyl ether and between about 0.8 and 60wt-% of propylene glycol phenyl ether.
 26. The composition of claim 21,wherein the biocidal composition further comprises between about 0.01and about 35 wt. % of an oxidizing agent, wherein the oxidizing agentcomprises hydrogen peroxide having an active concentration of 50% orgreater; and wherein said anionic surfactant is from about 5 to about 25wt-%.
 27. The composition of claim 21, wherein the biocidal compositionis diluted with water at a dilution ratio between about 5 parts waterper 1 part biocidal composition and about 128 parts water per 1 partbiocidal composition.
 28. The composition of claim 21, wherein thebiocidal composition is diluted with water at a dilution ratio betweenabout 16 parts water per 1 part biocidal composition and about 64 partswater per 1 part biocidal composition.
 29. The composition of claim 26,wherein hydrogen peroxide has an active concentration of 70% or greater;and wherein the composition does not include a peroxycarboxylic acid ora halogen.
 30. The composition of claim 27 wherein the anionicsurfactant is selected from the group consisting of (C₈-C₂₂)alkylsulfonate, linear alkyl benzene sulfonic acid, diphenylatedsulfonates, sulfonated oleic acid, and mixtures thereof and wherein thesparingly soluble and soluble organic solvents are selected from thegroup consisting of benzyl alcohol, diethylene glycol n-butyl ether,diethylene glycol n-propyl ether, diethylene glycol ethyl ether,diethylene glycol methyl ether, diethylene glycol t-butyl ether,dipropylene glycol n-butyl ether, dipropylene glycol methyl ether,dipropylene glycol ethyl ether, dipropylene glycol propyl ether,dipropylene glycol tert-butyl ether, ethylene glycol butyl ether,ethylene glycol propyl ether, ethylene glycol ethyl ether, ethyleneglycol methyl ether, ethylene glycol methyl ether acetate, propyleneglycol n-butyl ether, propylene glycol ethyl ether, propylene glycolmethyl ether, propylene glycol n-propyl ether, tripropylene glycolmethyl ether and tripropylene glycol n-butyl ether, ethylene glycolphenyl ether, propylene glycol phenyl ether and mixtures thereof. 31.The composition of claim 21 wherein soluble organic solvent and thesparingly soluble organic solvents are dipropylene glycol methyl etherand propylene glycol phenyl ether.
 32. The composition of claim 21,wherein the biocidal composition has sanitizing efficacy against abiofilm by penetrating the biofilm's extracellular matrix comprisingpolysaccharides.
 33. A method of manufacturing a biocidal compositioncomprising: combining from about 0.1 to about 2 wt-% of an anionicsurfactant, wherein the anionic surfactant is not a short chain (C1-C5)organocarboxylic acid and is a sulfonated acid; from about 0.1 to about2 wt-% of a soluble organic solvent in dilution; and from about 0.5 toabout 5 wt-% of a sparingly soluble organic solvent in dilution; whereinsaid sparingly soluble organic solvent is soluble in the soluble organicsolvent to an extent of about 15 wt-% or less; to form a biocidalcomposition; wherein the biocidal composition has a pH of less thanabout 3; wherein the biocidal composition provides a 3-log orderreduction of microbes on the biofilm-soiled surface; and wherein themicrobes comprise one or more of a Pseudomonas, an Escherichia, aStaphylococcus, and/or a Listeria.
 34. The method of claim 33 whereinthe anionic surfactant is selected from the group consisting of (C₈-C₂₂)alkylsulfonate, linear alkyl benzene sulfonic acid, diphenylatedsulfonates, sulfonated oleic acid, and mixtures thereof.
 35. The methodof claim 34 wherein the sparingly soluble organic solvent is selectedfrom the group consisting of benzyl alcohol, diethylene glycol n-butylether, diethylene glycol n-propyl ether, diethylene glycol ethyl ether,diethylene glycol methyl ether, diethylene glycol t-butyl ether,dipropylene glycol n-butyl ether, dipropylene glycol methyl ether,dipropylene glycol ethyl ether, dipropylene glycol propyl ether,dipropylene glycol tert-butyl ether, ethylene glycol butyl ether,ethylene glycol propyl ether, ethylene glycol ethyl ether, ethyleneglycol methyl ether, ethylene glycol methyl ether acetate, propyleneglycol n-butyl ether, propylene glycol ethyl ether, propylene glycolmethyl ether, propylene glycol n-propyl ether, tripropylene glycolmethyl ether and tripropylene glycol n-butyl ether, ethylene glycolphenyl ether, propylene glycol phenyl ether and mixtures thereof. 36.The method of claim 35 wherein the sparingly soluble and soluble organicsolvents are dipropylene glycol methyl ether and propylene glycol phenylether.
 37. The method of claim 36, wherein the method further comprisingcombining from about 0.01 to about 35 wt. % of an oxidizing agent,wherein the oxidizing agent comprises hydrogen peroxide having an activeconcentration of 70% or greater; and wherein the composition does notcontain a peroxycarboxylic acid or a halogen.
 38. The method of claim37, wherein the biocidal composition provides a 4-log order reduction ofmicrobes on the biofilm-soiled surface, and wherein the microbescomprise one or more of a Pseudomonas aeruginosa, a Staphylococcusepidermidis, a Staphylococcus aureus, a Listeria monocytogenes, and/oran Escherichia coli.
 39. The method of claim 38, wherein the biocidalcomposition has sanitizing efficacy against a biofilm by penetrating thebiofilm's extracellular matrix comprising polysaccharides.
 40. Themethod of claim 39, further comprising diluting the biocidal compositionwith water at a dilution ratio between about 5 parts water per 1 partbiocidal composition and about 128 parts water per 1 part biocidalcomposition.